Abstract
We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b (Itga2b) with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain (Il2rg) into intron 1 of Il2rg via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases.
Highlights
Allogeneic hematopoietic stem cell (HSC) transplantation is the firstline treatment in inherited hematopoietic disorders; the availability of human leukocyte antigen (HLA)-matched donors is limited.[1]
Ex vivo knockout of integrin alpha 2b (Itga2b) gene in bona fide HSCs It is known that stronger electroporation conditions to deliver CRISPR-associated protein 9 (Cas9)/ RNP are more efficient to edit genomes, but damage more cells, and that weaker conditions are gentler to cells but inefficient to edit
In this study, we demonstrated that Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockout and partial cDNA integration occurred in bona fide HSCs in a completely non-viral setting
Summary
Allogeneic hematopoietic stem cell (HSC) transplantation is the firstline treatment in inherited hematopoietic disorders; the availability of human leukocyte antigen (HLA)-matched donors is limited.[1]. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas[9] has opened new possibilities of site-specific insertion of therapeutic genes in human cells, including HSCs.[12,13] Previous studies have shown the feasibility of genome editing in HSCs to treat hematological disorders.[14,15,16] to realize the therapy, there are many issues to be addressed
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